Constitutive activating mutations of the human luteinizing hormone/chorionic gonadotropin receptor (hLHR) cause familial male-limited precocious puberty (FMPP), a non-central form of gonadotropin-independent precocious puberty. An activating mutation leads to elevated production of testosterone and results in precocious puberty. All the activating mutations of the hLHR resulting in FMPP identified by others as well as by our laboratory are located in the transmembrane of the hLHR. Transmembrane helix VI (TMVI) in particular is the hot-spot of mutation. Recently in a patient with FMPP we identified a novel heterozygous mutation A1738G which caused the replacement of Thr-580 by Ala. This mutation is downstream of the most frequently found activating mutation, Asp578Gly. Transient transfection and signal transduction via the cAMP pathway as well as the inositol phosphate pathway studies demonstrated that Thr580Ala did not display the activation phenotype. The influence of the ligands of other G-protein coupled receptors such as FSH and TSH were also found to have no effect on signaling in cells expressing this mutated receptor. The results of this study cautioned the interpretation of genotype-phenotype correlation in hLHR mutations and FMPP. In collaboration with Dr. Malcolm Martin and Dr. Ellen Leschek, we have identified two FMPP patients who developed testicular neoplasia. To study the potential tumorigenic effect of a constitutively activated LHR, we have generated an in vitro cell model and are in the process of generating a transgenic animal model. MA-10 cells were transfected with LHR carrying activating mutations. The profile of expressed genes in cells expressing the mutated LHR was compared with that of control cells using gene arrays on glass slide containing 22,000 mouse cDNAs. Preliminary studies of one mutated LHR indicated up-regulation of genes associated with cell proliferation and down-regulation of genes associated with differentiation. Interestingly, several genes known to be involved in spermatogenesis were also down-regulated in cells expressing the mutated LHR. Profile of gene expression affected by the more common activating mutation Asp578Gly will be compared with that affected by the somatic activating mutation Asp578His found in some patients with testicular tumor. The antithesis of FMPP is Leydig Cell Hypoplasia (LCH). In LCH patients, mutation inactivates the LHR resulting in reduced production of testosterone causing hypergonadotrophic hypogonadism or male pseudohermaphroditism. A novel missense mutation A340T resulting in the substitution of Ile-114 by Phe, which affects one of the six Leu repeats in the extracellular domain of hLHR receptor, has been identified in a patient with LCH. The mutant receptor failed to trigger cAMP production upon hCG stimulation in transient expression study. It is known that the mutated LHR, be it activated or inactivated, are abnormally processed by cells. To investigate the trafficking of mutated LHR in vitro, we have fused the coding sequence of Green Fluorescent Protein (GFP) to that of wild- type and mutated LHR. The trafficking of the fused protein was studied by fluorescent microscopy. Information generated should further our understanding of the cellular processing of the LHR. The impact of activating mutation of the LHR has always been considered to be limited to sexual development of the patient. The abnormal social behavior of the patient was thought to be secondary to precocious sexual maturation. Expression of the LHR in the brain had been demonstrated. We speculate that the abnormal behavior of FMPP patients is caused by the expression of the mutated LHR in the brain. The first step to examine this hypothesis is to identify the cellular location of the LHR in the brain. Transgene cassettes, with an open reading frame, encoding a fusion protein of enhanced green fluorescent protein (EGFP) and hLHR mutant with substitution Asp578Gly or Asp578His, were generated. The expression of the fusion protein is driven by a 2.1 kb mouse LHR 5!| flanking sequence. The transgene cassettes will be used to generate transgenic mice. The animal model generated will be used to study the impact of constitutively activated LHR on spermatogenesis as well as sexual and neurological development.